Small, non-coding, regulatory RNA species such as microRNAs have emerged in recent years as a powerful agent in regulating gene expression in eukaryotic cells. First discovered in 1993 (Lee at al., Cell 75: 843-854 (1993)), microRNAs are an abundant new class of regulatory elements that have been shown to impact all aspects of normal cellular processes in both plants and animals, including cell death, differentiation, and proliferation, as well as abnormal processes including cancer (Bartel, Cell 116: 281-297 (2004); Du and Zamore, Development 132: 4645-4652 (2005); Pillai, RNA 11: 1753-1761 (2006)). In general, an miRNA is composed of a highly conserved core sequence of 21-23 nucleotides (the mature miRNA) contained within a less well conserved precursor sequence (pre-miRNA) ranging in size from 60 nucleotides to more than 120 nucleotides. This pre-miRNA sequence is part of a larger primary transcript that may contain a single pre-miRNA or two or more pre-miRNAs arranged as paired or polycistronic transcripts. MicroRNA expression has been found to be highly specific and, in many cases, sequestered by tissue type and/or developmental stage. For this reason, discovery of new microRNAs requires the cloning of RNA species that may be expressed only in certain cells harvested at particular times. The availability of a generally applicable and efficient cloning method is, therefore, key in advancing knowledge of both the number of microRNAs present in a given genome and their specific role in that organism's cells.
Since 2001, several methods for cloning microRNAs and other small RNA species from total cellular RNA have been advanced (Berezikov et al., 2006; Cummins et al., 2006; Elbashir et al., Genes and Development 15: 188-200 (2001); Lau et al., Science 294: 858-862 (2001); Pfeffer et al., Current Protocols in Molecular Biology, 26.4.1-26.4.18 (2003); Sunkar and Zhu, The Plant Cell 16: 2001-2019 (2004)). Cloning small RNAs generally begins with the isolation and purification of total cellular RNA from a relevant cell source. More recent variations on the basic scheme advocate enriching the RNA target pool for species in the proper size range. This entails taking the total cellular RNA pool and isolating only those RNAs that fall below or within a certain size range. Commercial products are available to remove larger RNA species from the target pool that would compete in the subsequent process that forms the substance of the described method.
Once an enhanced RNA target pool has been purified, by whatever means, the next step is to attach a 3′-end blocked linking group that will ligate to the 3′-end of the small RNA species. Generally, a 5′-end linking group is also ligated to the small RNA species. Then reverse transcriptase polymerase chain reaction (RT-PCR) is performed wherein the resulting fragments are cloned into vectors to create a cDNA library comprising a heterogeneous collection of small cellular RNA species. The cloned fragments can then be sequenced and analyzed to determine the identity and genomic origin of the small RNA species present in the sample.
Current methods have enabled investigators to identify hundreds of unique miRNAs, and there are estimates that thousands of unique miRNAs may exist. Additionally, several new classes of small, regulatory RNAs have been discovered in the past few years. These new classes include endogenous silencing RNAs (endo siRNAs), PIWI-interacting RNAs (piRNAs), 21U RNAs, and repeat-associated siRNAs (rasiRNAs), all of which have been discovered by direct cloning from specific RNA sources. Production of small RNA libraries is a complex task and it is possible to produce libraries that are incomplete or contain skewed subsets of the RNA species present in the original sample. There is a need for more efficient methods of small RNA cloning that can be used by less-skilled technicians to clone and identify miRNAs.